The aa3-type cytochrome c oxidase of Rhodobacter sphaeroides is an excellent model of the mammalian mitochondrial oxidase, and a system has been prepared for rapid site-directed mutagenesis and over-expression of mutant oxidases in Rb. sphaeroides. Recent high resolution structures of cytochrome oxidase provide the information needed to explore the roles of subunit III in oxidase function at the molecular level. Subunit III is one of the three highly conserved core subunits of cytochrome oxidase, but its role in oxidase function is far from clear. Subunit III has a unique structure that includes 3 tightly bound phospholipids that interact with residues of both subunits III and I. Our preliminary data show that subunit III is necessary for the assembly of the heme a3-CuB active site in subunit I, and prevents the assembled oxidase from rapidly inactivating during catalysis. Suicide- inactivation involves a structural change that eliminates the ability of heme a3 to bind CO. Previous reports and the new structures suggest that subunit III also modulates the efficiency of proton pumping by cytochrome oxidase, binds ADP/ATP at a site and that could directly influence the activity of the pump, and provides a path, via the lipid pool, for 02 to access the buried heme a3-CuB site. To explore the molecular mechanisms of oxidase assembly, suicide inactivation, pumping efficiency, nucleotide binding, and 02 delivery, site directed mutants of key residues and oxidases lacking subunit III have been prepared. In addition, four mutations in subunit III found to cause mitochondrial diseases in humans have been introduced into the Rb sphaeroides oxidase. The mutant oxidases will be assayed for alterations in expression, activity, the structure of the redox-active metal centers, internal electron transfer, CO, cytochrome c and nucleotide binding, and proton pumping efficiency. The goals are to 1) define the roles of subunit III in oxidase function, 2) define the mechanism of suicide-inactivation, 3) define the functional defects in cytochrome oxidase that cause the mitochondrial diseases, and 4) use the interaction of subunit III with the heme a3-CuB active site to further explore the mechanisms of energy coupling by cytochrome oxidase.